Table of Contents
- Northern Blot Definition
- Principle of Northern Blot
- Materials Required for Northern Blot
- Procedure/Steps of Northern Blot
- Result Interpretation of Northern Blot
- Applications of Northern Blot
- Limitations of Northern Blot
- References
Northern Blot Definition
- Northern blot is a laboratory technique based on the principle of blotting, designed for analyzing specific RNA molecules within a complex mixture.
- It is a modified form of the Southern blotting technique, which was originally developed for analyzing DNA sequences.
- In molecular biology, detecting specific nucleic acid sequences from various biological samples is crucial, making blotting techniques highly important in this field.
- The fundamental principle of Northern blotting is the same as that of Southern blotting, except that the probes used in Northern blotting are specific to RNA sequences rather than DNA.
- This technique provides valuable information about the length of RNA sequences and helps identify any variations present in those sequences.
- Although primarily used for identifying RNA sequences, Northern blotting can also be applied for quantifying RNA levels within samples.
- Since its development, several modifications of the technique have been introduced to allow the analysis of different RNA types, including mRNAs, pre-mRNAs, and short RNAs.
- For many years, Northern blotting served as the main technique for studying RNA fragments; however, with time, newer, faster, and more cost-effective methods such as RT-PCR have gradually replaced it.
Principle of Northern Blot
- The principle of Northern blotting is similar to other blotting techniques, relying on the transfer of biomolecules from one membrane to another for analysis.
- In this method, RNA samples are separated according to their size using gel electrophoresis.
- Because RNA molecules are single-stranded, they can form secondary structures through intermolecular base pairing; therefore, electrophoresis is carried out under denaturing conditions to prevent such structures.
- After separation, the RNA fragments are transferred from the gel onto a nylon membrane, as nitrocellulose membranes are ineffective for RNA binding.
- Once transferred, the RNA segments are immobilized on the membrane using fixing agents to ensure they remain in place during further analysis.
- Detection of specific RNA fragments is achieved by adding a labeled probe that is complementary to the RNA sequences bound on the membrane.
- The process of hybridization between the labeled probe and the target RNA sequence provides the basis for specific and accurate detection.
- Since Northern blotting separates RNA fragments by size, it allows the determination of transcript sizes in addition to identifying specific RNA molecules.
Materials Required for Northern Blot
Equipment required for Northern blot:
- Agarose gel cast
- Power supply
- Microwave
- Centrifuge
- Heating block
- UV crosslinker
- Hybridization oven
- Hybridization vessels
- Vials
- Forceps
- Pipettes
- Glass tubes
Materials and reagents required for Northern blot:
- Agarose gel
- Sodium citrate
- Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate
- Sodium hydroxide (NaOH)
- Hydrochloric acid (HCl)
- Formaldehyde
- Glycerol
- Ethidium bromide
- Bromophenol Blue
- RNA ladder
- Magnesium chloride (MgCl₂)
- Sodium chloride (NaCl)
- Polyvinylpyrrolidone (PVP)
- Bovine Serum Albumin (BSA)
- Sodium dodecyl sulfate (SDS)
- Sodium dihydrogen phosphate (NaH₂PO₄)
- Tris-HCl buffer
- Triton X
- Dithiothreitol (DTT)
- Taq buffer
- Taq polymerase
Procedure/Steps of Northern Blot
a. Separation of RNA on a denaturing gel:
- Prepare the RNA gel solution by adding formaldehyde to the agarose solution to create a denaturing environment.
- Assemble the gel cast and pour the prepared denaturing gel into it. Insert a comb with suitable teeth to form wells as the gel sets.
- Once the gel has solidified, remove the comb and equilibrate the gel with running buffer for about 30 minutes before electrophoresis.
- Mix 15 µg of RNA sample with an equal volume of RNA loading buffer, and prepare 3 µg of RNA markers in the same buffer.
- Incubate all samples at 65°C on a heating block for 12–15 minutes.
- Load the RNA samples into the wells of the equilibrated gel, with the RNA markers added to the first row of wells.
- Run the gel electrophoresis at 125V for approximately 3 hours to separate RNA fragments based on size.
b. Transfer of RNA from gel to nylon membrane:
- Cut a nylon membrane slightly larger than the gel size and prepare a filter paper of the same dimensions.
- After electrophoresis, carefully remove the RNA gel from the tank and rinse it with water.
- Place a slightly larger oblong sponge in a glass dish and fill the dish with SSC buffer until the sponge is half-submerged.
- Lay a few pieces of Whatman 3mm filter paper on top of the sponge and wet them with SSC buffer.
- Place the RNA gel on top of the wetted filter paper and roll a glass pipette over it to remove air bubbles.
- Wet the nylon membrane in distilled water on an RNase-free dish for about 5 minutes.
- Carefully place the wetted membrane on top of the gel, ensuring no air bubbles form between the layers.
- Flood the surface with SSC buffer and place additional filter papers on top of the membrane.
- Add a glass plate on top to keep the setup in position and leave it overnight for efficient RNA transfer.
c. Immobilization:
- After transfer, remove the gel, rinse the membrane with SSC, and allow it to dry.
- Sandwich the membrane between two pieces of filter paper and bake it in a vacuum oven at 80°C for 2 hours to fix the RNA.
- Alternatively, wrap the membrane in UV-transparent plastic and expose it to UV radiation for an appropriate time using a UV transilluminator to immobilize RNA.
d. Hybridization:
- Prepare DNA or RNA probes labeled to a specific activity of greater than 10⁸ dpm/µg, and remove any unincorporated nucleotides.
- Moisten the membrane carrying immobilized RNA with SSC buffer.
- Place the membrane in a hybridization tube with the RNA side facing up, then add 1 ml of formaldehyde solution.
- Incubate the tube in a hybridization oven at 42°C for 3 hours.
- If a double-stranded probe is used, denature it by heating for 10 minutes at 100°C before use.
- Add the required volume of the denatured probe into the hybridization tube and continue incubation at 42°C.
- After incubation, pour off the hybridization solution and wash the membrane with a suitable wash solution to remove unbound probes.
- Finally, visualize the hybridized RNA fragments by observing the membrane under autoradiography.
Result Interpretation of Northern Blot
- The results of Northern blotting are visualized under autoradiography, where RNA appears as distinct bands on the membrane.
- Each band represents a specific RNA fragment that has hybridized with the labeled probe.
- By comparing the position of these bands with those of the RNA markers or ladder, the approximate length of the RNA fragments can be determined.
- The intensity of the bands provides a semi-quantitative measure of RNA abundance, indicating relative expression levels of the target RNA among different samples.
Applications of Northern Blot
- Northern blotting is used for the identification and separation of RNA fragments obtained from various biological sources.
- It serves as a sensitive method for detecting the transcription of DNA fragments that may later be used as probes in Southern blotting.
- The technique enables the detection and quantification of specific mRNA molecules from different tissues and organisms.
- It is widely applied in gene expression studies, particularly for investigating the overexpression of cancer-associated genes and monitoring gene expression during transplant rejection.
- Northern blotting has been utilized as a molecular diagnostic tool for certain diseases, including Crohn’s disease.
- The method is also employed for detecting viral microRNAs, which play key roles in the regulation of viral infections.
Limitations of Northern Blot
- Northern blotting has relatively low sensitivity compared to modern molecular techniques such as RT-PCR and nuclease protection assays.
- It requires a large quantity of RNA sample, which must be of high purity and integrity for accurate results.
- The procedure is time-consuming and technically complex, particularly when multiple probes are used for detecting different RNA targets.
References
- He, S. L., & Green, R. (2013). Northern blotting. Methods in Enzymology, 530, 75–87. https://doi.org/10.1016/B978-0-12-420037-1.00003-8
- Josefsen, K., & Nielsen, H. (2011). Northern blotting analysis. In Methods in Molecular Biology (Vol. 703, pp. 87–105). https://doi.org/10.1007/978-1-59745-248-9_7
- Blevins, T. (2010). Northern blotting techniques for small RNAs. In Methods in Molecular Biology (Vol. 631, pp. 87–107). https://doi.org/10.1007/978-1-60761-646-7_9
- Mishima, E., et al. (2015). Immuno-Northern blotting: Detection of RNA modifications using antibodies against modified nucleosides. PLoS ONE, 10(11), e0143756. https://doi.org/10.1371/journal.pone.0143756
- Koscianska, E., et al. (2011). Northern blotting analysis of microRNAs, their precursors, and RNA interference triggers. BMC Molecular Biology, 12, 14. https://doi.org/10.1186/1471-2199-12-14
- Brown, T., & Mackey, K. (2001). Analysis of RNA by Northern blot hybridization. Current Protocols in Human Genetics, Appendix 3, Appendix 3K. https://doi.org/10.1002/0471142905.hga03ks30
